Cell-free protein expression provides a simple and efficient method for generating proteins without the complications of cell culture, cell engineering, or cell transfection. Cell-free systems for expressing recombinant proteins address various limitations of cell-based expression systems such as protein toxicity, protein degradation, protein aggregation and misfolding, uncontrolled post-translational modification, or negative effects of protein expression on cell growth due to sequestration of cellular machinery. Significantly higher quantities of proteins can be expressed in a shorter period of time using a cell-free protein expression system that can be employed for downstream high-throughput structural and functional analyses. Such in vitro protein expression also has significant advantages in terms of cost savings, streamlined production, easier scale-up, and simplified purification. In a cell-free protein expression system, a desired protein of interest is expressed by adding a DNA or RNA that encodes a gene of the protein of interest to a transcription-translation-competent cellular extract, and performing the transcription and/or translation of the gene of interest. The transcription and translation may be coupled in a single reaction to enable immediate translation of a newly synthesized mRNA into protein (coupled in vitro transcription-translation system or coupled transcription-translation in a cell-free system). The coupled in vitro transcription and translation generally increases the yield of expressed proteins with less time and in vitro manipulation. The immediate translation of the mRNA avoids possible adverse effects associated with mRNA degradation or misfolding.
One limitation of in vitro transcription-translation systems is that they require large quantities (generally in microgram quantities) of a DNA template. Generally, sufficient amounts of DNA can be obtained through multiple workflow steps and significant labor effort, for example, by cloning the DNA into a plasmid vector and propagating the plasmid in a host cell (e.g., E. coli) or by synthesizing DNA from multiple polymerase chain reactions (PCR). However, PCR is often not amenable for large-scale generation of high-quality DNA, due in part to the high mutation rate of PCR. Additionally, the thermal cycling of PCR reactions is difficult to scale-up to larger reactions due to limitations on how quickly temperatures can be ramped in large volumes. Moreover, PCR products, being linear DNA sequences, may be rapidly degraded by the action of nucleases that are present in cell-free transcription-translation extracts. Further, sub-cloning of a gene of interest into a plasmid vector followed by high-scale propagation in E. coli through genetic selection is time-consuming and labor intensive.
Isothermal DNA amplification techniques such as rolling circle amplification (RCA) can be employed to generate large quantities of high-quality DNA with less effort, time, and expense, starting from a circular nucleic acid template. Rolling circle amplification reactions are isothermal, making scale-up to larger reaction sizes straightforward as there is no requirement for rapid heating and cooling. Rolling circle amplification generates RCA products that are tandem repeat units (concatamers) of the template nucleic acid sequence. RCA of a plasmid DNA, followed by coupled in vitro transcription and translation, is possible to generate the protein of interest. However, these plasmids are created via standard cloning methods involving genetic-selection inside a host cell such as E. coli. Such plasmids therefore contain many additional coding and non-coding sequences including sequences for the origin of replication (for example, oriC), antibiotic selection (for example, amp for beta-lactamase), and accessory sequences that are used for selection and/or screening plasmids in the host cells, such as lacZ, beta-galactosidase. Transcription and/or expression of these ancillary sequences are not desired, or may be considered inefficient, relative to the gene of interest that is meticulously sub-cloned into the plasmid. Consequently, PCR amplification of a gene of interest within the plasmid is often employed for cell-free protein expression.
There exists a need for improved in vitro transcription and translation systems for easy generation of desired proteins that are transcribed and translated from a DNA that is optimally free of any of the extraneous sequences and host cell contaminants, and does not require PCR synthesis. Also, it is desirable to increase the yield of cell-free protein systems using methods that are simplified and less time-consuming.